The infectious disease that threatens health of one third of the world's population is tuberculosis (TB). Despite the presence of anti-tuberculosis drugs, about 1% of the world's population contracts tuberculosis annually and 1.3 million people die from TB-Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are gradually increasing due to poor managing the TB patients or TB suspicious groups. In recent years, the first case of totally drug resistant TB (TDR-TB) has been reported in India. To reduce transmission of TB and improve outcomes for TB patients, a rapid and accurate drug treatment after drug susceptibility test (DST) is necessary. Diagnosis of the resistance of TB to certain drugs among standard first-line anti-TB agents is very important in identifying MDR-TB, XDR-TB, and TDR-TB and preventing ineffective drug therapies.
General DST methods are based on culturing cells in solid and liquid media. According to solid medium culture methods, Mycobacterium tuberculosis (MTB) is inoculated into a solid medium supplemented with an antibiotic and the formation of colonies is observed by naked-eye detection. Since MTB grows slowly (the cells differentiate for about 24 hours), conventional DST methods take at least 4 to 6 weeks. The use of liquid media accelerates the growth of MTB to reduce the time required for DST. A commercial DST platform using liquid media called MGIT 960 system, is available. The MGIT system detects a fluorescence signals when MTB grows and consumes oxygen. The DST time of this MGIT system is as short as approximately 1 week. However, this method causes an increase in error rate because the growth of MTB cells is not directly observed but is indirectly identified.
A microscopic-observation drug susceptibility (MODS) method for direct observation was developed to reduce DST time. According to this method, a sputum sample together with a drug is inoculated into a 24-well plate and colony formation is monitored daily, which plays an important role in identifying whether MTB grows. This method can yield DST results within one week. However, single cells of MTB cannot be immobilized in liquid media, making it impossible to track the single cells. To obtain DST results, a microscopic observation over the entire well area is essential for the detection of colony formation and a long-term observation is inevitable.
Many lab-on-a-chip technologies have been developed for single cell tracking: an array of micro-scale wells, passive microfluidic trapping, an actively valved microfluidic system, and droplet microfluidics. In the array method using micro-scale wells and passive microfluidic trapping, the cells settle via gravity and are trapped in the weirs by fluid flow. However, the difficulty associated with controlling the fluidic environment hinders multiplex assays for drug testing. To better control cell and drug loading, actively valved microfluidics employs a computer-controlled, pneumatically actuated trapping method to precisely control small quantities of liquids via multiple control elements. However, this approach relies on complex control, which is associated with high costs and excessive operating efforts. Therefore, this method is not applicable for routine clinical drug testing. Droplet microfluidics employs a small number of chambers for multiplexing drug tests. However, these methods do not ensure the immobilization of bacterial cells in the liquid, which is necessary for single cell tracking.
Inoculum effect (IE) is an unwanted phenomenon in laboratory and may lead to overestimation of in vitro resistance, causing an increase in minimal inhibitory concentration (MIC). Consequently, inoculum effect deteriorates the reproducibility of experiments. Furthermore, since MIC is a parameter determining the microbiological efficacy of antibiotics in pharmacokinetics, different MIC values by cell densities may limit the clinical evaluation of antibiotics.